The present invention relates to recombinant DNA molecules encoding plasmid DNA replication origins in Thermus, as well as to shuttle vectors which contain the same.
Many species of bacteria contain small circular extrachromosomal genetic elements, known as plasmids. Plasmids have been found in a number of bacteria which live in extreme environments, including the thermophiles, which live at high temperatures of more than 55.degree. C. (Munster et al., Appl. Environ. Microbiol. 50:1325-1327 (1985); Kristjansson and Stetter, in `Thermophilic Bacteria`, Kristjansson, ed., p. 1-18 (1992)). However, most thermophile plasmids remain `cryptic` in that functional genes have not been isolated from them, hence leaving their functional significance speculative (Hishinuma et al., J. Gen. Microbiol. 104:193-199 (1978); Eberhard et al., Plasmid 6:1-6 (1981); Vasquez et al., FEBS Lett. 158:339-342 (1983)). Common genes found in plasmids include those encoding plasmid replication and cellular maintenance, antibiotic resistance, bacteriocin production, sex determination, and other cellular functions (Kornberg and Baker, `DNA Replication`, 2.sup.nd ed. (1991)).
It is often particularly difficult to cultivate thermophilic bacteria within the laboratory. They require high temperatures and often-unknown environmental conditions for acceptable growth (Kristjansson and Stetter, in `Thermophilic Bacteria`, Kristjansson, ed., p. 1-18 (1992)). However, with the advent of genetic engineering, it is now possible to clone genes from thermophiles into more easily cultivatable laboratory organisms, such as E. coli (Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). The expression of such genes can be finely controlled within E. coli.
A Thermus-E. coli shuttle vector would be desirable if one needs to have the convenience of cloning in E. coli, isolation of DNA from E. coli for further manipulations and subsequently gene selection and expression in Thermus. Such Thermus-E. coli shuttle vectors could be used to screen, select and express thermostable proteins in Thermus. Using these vectors, a gene could, for example, be mutated within a mesophile, transferred to a thermophile, and then its encoded protein selected for increased thermostability. In this way, mesophile-thermophile shuttle-vectors can be used to conduct directed evolution, or protein engineering, on desirable gene products.
There is commercial incentive to produce thermostable proteins which are usually more thermostable in denaturing conditions then mesophilic counterparts (Wiegel and Ljungdahl, CRC Crit. Rev. Biotech. 3:39-108 (1984); Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). These thermostable enzymes can also be used in a variety of assays, such as PCR, restriction enzyme-mediated PCR, thermo-cycle DNA sequencing and strand-displacement amplification, in which high temperatures are desirable. The shuttle vectors of the present invention should facilitate production of such thermostable proteins.